Using intravital 2-photon imaging methods developed in the LBS over the past several years, we are now able to routinely image peripheral organs and tissues so that immune effector cell behavior and to some extent effector functions during infectious processes can be observed. Using these new methods, we previously described lymphoid and myeloid cell dynamics in BCG-induced granulomas in the liver and showed that only a small fraction of the antigen-specific T cells within a granuloma undergo migration arrest at any time. This small fraction of such arrested cells correlates quantitatively with the fraction of specific cells making the key effector cytokine IFNgamma. These findings provided entirely new insights into the way in which effector T cells operate in the natural in vivo setting and point to the large differences between in vitro evoked responses and the actual behavior of effector cells at sites of infection. Many of these observations have been repeated in M tuberculosis-infected animals. The finding that only a small fraction of the cells being imaged in a tissue are actually engaged in effector function at any time and that these have a dynamic behavior distinct from the bulk of the imaged cells raises critical questions about many existing and ongoing studies in other laboratories using intravital 2-photon imaging, in which the bulk or average behavior of clonally-related cells is taken as representative of the functional population. In addition to the liver granuloma model, we have published studies of T-cell motility in infected lungs, comparing the response of CD4+ T cells to influenza vs. BCG infection and addressing the question of whether in this site, the same limited activation of effector function is seen as in the liver and whether this varies with the pathogen. These studies confirmed the conclusions of our liver granuloma model, in that only a very small fraction of the mycobacteria-specific T cells arrest movement and produce cytokine (IFN-gamma) in this infected tissue setting. In striking contrast, the influenza antigen-specific T cells showed much greater fractional migration arrest and a correspondingly greater fraction of cytokine producing T-cells. These latter results emphasize that the extent of the effector response varies greatly depending on the microbe, in apparent concordance with the density of presented antigen (influenza >> mycobacteria). In other studies, we are examining how the structure of lymph nodes is organized to limit access of invading organisms to the blood stream. These studies showed a crucial role of CD169+ subcapsular sinus macrophages in responding to lymph borne pathogens by producing the cytokine IL-18, which together with others such as IL-12 or type 1 interferon, resulted in rapid cytokine-induced cytokine production by a diverse set of lymphocytes (memory CD8 T cells, gamma-delta T cells, NK cells, and NKT cells). IFNgamma plays a critical role in preventing replication of incoming bacteria in the lymph node and in preventing systemic dissemination. Remarkably, these various lymphocytes population, while highly motile, show constrained localization to the regions near to the subcapsular sinus-lining CD169+ macrophages, revealing a very specific tissue micro-anatomy that supports robust innate responses to incoming pathogens to present their dissemination. More recently we have begun exploration of the interaction of microbes with the host gut, in particular, examining how potential pathobionts are constrained to exist in a commensal-like homeostatic state with the host through the action of a combination on innate lymphocytes (ILC) and T cells. Combining our new static imaging tools with the use of various reporter and genetic engineered strains of mice, we have been able to quantify the extent of cytokine signaling of epithelial cells in the small bowel resulting from IL-23 activation of IL-22 production from ILC3 based on pSTAT3 presence and to determine that these ILC operate in conjunction with Th17 and Treg to shape the normal commensal microbiota. During the past year, we have extended these studies, revealing that ILC3s and adaptive CD4 T cells participate sequentially to establish the mature state of non-inflammatory commensalism with certain bacteria. Strikingly, the ILC3 and CD4+ T cells use different mechanisms in regulating bacterial numbers, as evidenced by a lack in wild-type mice of the extensive pSTAT3 signature seen in RAG mice. This raises doubts about the usual assumption that ILC subsets and CD4 effector subsets share effector mechanisms but differ only in whether an antigen-specific receptor is used to drive activation. The imaging methods developed for this project have also been employed to discover the site of IL-25 action and to detail the proliferation and migration of activated ILC2 from gut to peripheral organs where they mediate anti-helminth protective immunity (see AI000545-28). These studies show that in contrast to the usual assumption that ILC are tissue resident effector cells, they have a similar behavior to adaptive T cells in that they are activated, proliferate, and differentiate in one tissue (in the case of iILCs, the small bowel lamina propria), the migrate into lymph and then the blood circulation in an S1P-dependent manner to exit at a distant tissue to mediate protective function. We have also initiated studies examining the cellular basis for strong host protection against malaria infection in a mouse model using a push-pull vaccination scheme developed by a joint graduate student shared with the laboratory of Prof. Adrian Hill at University of Oxford. These studies have revealed a key role for CD8 T cells with a tissue resident memory cells phenotype in terms of CD69 expression along with chemokine receptor expression consistent with liver homing. We are in the midst of examining how they patrol and detect the liver stage parasite, and how they mediate elimination of the infected cells once detected, using a combination of intravital dynamic imaging and Histo-cytometry. In collaborative studies in a mouse model of LCMV and human studies in HIV infected subjects, we have collaborated using our histo-cytometry methods to reveal the localization of cells that are able to expand and show strong effector function after exhaustion due to chronic antigen exposure, as well as to localize potentially important effector CD8 T cells within lymph node structures in infected individuals. Finally, we have returned to the study of lethal influenza infection. Using data from our previous systems-level analysis, we examined whether tissue based RNA expression data could inform a blood signature that would allow prediction of lethal infection at an early stage. While we could validate the specificity of the lethal vs. influenza infection pan signature, we could not reliably predict lethality at one LD50 in infected mice. We are presently exploring whether this is due to small variations in viral exposure, or more interestingly, if this reflects somatic variations that influence the pace of the protective adaptive immune response. We have also developed a 12-color staining panel to explore the distribution of immune and tissue cells in large volumes of the mouse lung, using a combination of our Ce3D clearing method and histo-cytometry.